Cell lines and tissue culture
All cell lines were obtained from ATCC and grown in monolayer at 37°C/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) or Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum, 0.1mg/mL penicillin, 100 units/mL streptomycin, and 15mM Hepes. Generation and characterization of B3-KO and isogenic CRISPR-control cell lines SW756 and HT3 was described previously11. In brief, a single-vector lentiviral system was used, driving expression of Cas9 and the gRNA sequence. KO was confirmed by sequencing of the SERPINB3 gene, Western blot showing no protein product, and sequencing of most-likely off-target genes to confirm specificity. For the current paper, SiHa and C33A cell lines were engineered to stably express pULTRA (Addgene 24129) mammalian vector driving expression of the wild type SERPINB3 gene, or SERPINB3 mutant encoding a single alanine to arginine substitution at amino acid 341 (B3-P14m).
Irradiation of cell lines and mice
Cells in dishes were irradiated at a dose-rate of ~ 300cGy per minute using the RS-2000 Biological System (Rad Source, Suwanee, GA), calibrated once monthly by a medical physicist. Sham irradiation conditions were transported to the irradiator and placed at room temperature conditions while delivering radiation. Mouse xenograft tumors were irradiated using the Xstrahl Small Animal Radiation Research Platform (SARRP) 200 (Xstrahl Life Sciences, Suwannee, GA). After being fitted with a nose cone, mice were individually subjected to isoflurane anesthesia and imaged by on-board micro-computed tomography (CT). CT images imported into Muriplan were used to select an isocenter. The tumor was then irradiated using anterior-posterior opposed beams using the 10 mm × 10 mm collimator at a dose rate of 3.9 Gy/min. Sham irradiated mice were transported to the SARPP facility and remained in the room at the same conditions during radiation of the irradiated mice.
Cell cycle analysis
Cells were seeded at 0.15x10^6 cells per well and treated 24 hours later with 4Gy or Sham RT. Cells were fixed and permeabilized with Triton X-100 48 hours later (after optimization of IR dose and incubation time), stained with propidium iodide, and cell cycle distribution determined using flow cytometry of DNA content. FlowJo™ Software (Becton, Dickinson and Company, Ashland, OR, USA) was used to analyze and visualize the data.
Gamma-H2AX foci formation assay
Cells were seeded in 4-well chamber slides (Nunc® Lab-Tek® II Chamber Slide™, C6807, Millipore Sigma, St Louis, MO, USA) to 50–60% confluence and allowed to adhere to the slide for 24 hours. Cells were then treated with sham or 2Gy radiation as described in the “Irradiation of cell lines and mice” section and incubated at 37˚C for 30 minutes or 24 hours prior to washing with PBS and fixation with 3% PFA. Cells were then permeabilized with 0.1% Triton-X and washed with PBS before blocking with blocking buffer (10% FBS, 0.5% BSA in PBS) for one hour at room temperature. Slides were incubated at 4˚C overnight with 1:300 anti-gH2AX antibody (05-636, Millipore Sigma, St Louis, MO, USA). Slides were washed in three changes of PBS and incubated with 1:500 anti-mouse-IgG AlexaFluor 488 conjugated secondary antibody for 3 hours at room temperature. Slides were mounted with VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI (H-1500, Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained on the Zeiss LSM510. Images were then analyzed using ImageJ Software to quantitate foci per nucleus for a random selection of six 100X fields of view, corresponding to 30–50 cells per condition.
Cell death assay
Cells were seeded in order to achieve 50–70% confluence 24 hours later in a 96-well glass-bottom, opaque-walled dish (Greiner Bio-One™ CellStar™ µClear™ 96-Well, Cell Culture-Treated, Flat-Bottom Microplate, Fisher, 7000166). 24 hours after plating, plates were treated with varying doses of IR or Sham, with one-hour pre-incubation of inhibitors where indicated. Cisplatin was added 1 hour prior to radiation, where indicated, at the half-maximal inhibitory concentration (IC50) of cisplatin monotherapy determined by Alamar blue assay (0.5µM for SW756 and 0.01µM for HT3). At established time points, Sytox™ orange (1:30,000, Fisher Scientific, S11368) and Hoescht 33342 (1:2,000) were added 15–30 minutes prior to quantitative imaging on the Cytation™ 5 multi-mode reader (BioTek® Instruments, Inc., Winooski, Vermont). Percent dead cells was determined by optimized automated counting of Sytox-positive cells divided by Hoescht-positive nuclei, averaged over triplicate wells.
Cells were seeded and treated as in the cell death assay methods, except scaled to 6-well or 10cm dishes in order to obtain adequate cell lysate. At established time points, coinciding with the cell death assay, cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) supplemented with proteinase/phosphatase inhibitors and PMSF. Equal parts protein (25–30µg) and Laemmli sample buffer (Santa Cruz Biotechnology, Dallas TX) were boiled at 95 degrees Celsius for 10 minutes and gel electrophoresed on 4–20% gradient gels (Mini-Protean TGX, Bio-Rad, Hercules, CA), transferred to PVDF blot using the Trans-Blot Turbo Transfer system (Bio-Rad, Hercules, CA), blocked with 5% milk:TBS-Tween and incubated with 1:4,000 anti-SCCA1 antibody (NBP2, Novus International, Saint Louis, MO) overnight at four degrees Celsius, 1:100,000 anti-Actin (A5441, Santa Cruz Biotechnology, Dallas, TX), 1:2,000 anti-GAPDH-HRP conjugated antibody (D16H11, Cell Signaling Technology, Danvers, MA) for 2 hours at room temperature, 1:1000 anti-GSMDC1 (NBP2-33422, Novus Biologicals, LLC, Littleton, CO) overnight at four degrees Celsius, 1:1000 anti-p-MLKL (91689S, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-Cleaved Caspase3 (9661, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius,, 1:1000 anti-Cleaved Caspase7 (8438, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-Cleaved PARP (5625s, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-Full Length PARP (9524s, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-Caspase7 (12827, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:500 anti-Caspase3 (sc-7272, Santa Cruz biotechnology, INC, Texas) overnight at four degrees Celsius, 1:1000 anti-RIP3 (57220, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-RIPK3 (ser227) (93654s, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:1000 anti-RIPK1 (ser166) (44590, Cell Signaling Technology, Danvers, MA) overnight at four degrees Celsius, 1:10000 anti-p-ATM (ab81292-100UL, Abcam, Cambridge, MA) overnight at four degrees Celsius, 1:1000 anti-ATM (NB100-309, Novus Biologicals, LLC, Littleton, CO) overnight at four degrees Celsius. Anti-mouse or anti-rabbit HRP-conjugated secondary antibody was used for detection with ECL chemiluminescent reagent (GE Healthcare Life Sciences, Pittsburgh, PA), visualized, and quantified using the Bio-Rad ChemiDoc MP imaging system and Image Lab software (Bio-Rad, Hercules, CA).
Clonogenic cell survival assay
500–1000 cells per well were seeded in 6-well plates 24 hours prior to treatment with increasing doses of radiation (2, 4, 6 Gy x 1) and incubated for 1–3 weeks until control plates formed visible colonies (≥ 50 cells). IC50 concentration of cisplatin was added 1 hour prior to radiation, where indicated. Plates were fixed and stained with 0.5% Crystal Violet, 30% Methanol, 10% Acetic Acid, 60% ddH2O for 30 minutes, rinsed in tap water and air dried at room temperature. Surviving fraction was calculated as the number of colonies ÷ (500 * plating efficiency) and plotted on a log10 scale as per convention. The linear quadratic equation was fit to each dataset using GraphPad Prism 8©. The dose modifying factor (DMF) was determined as the ratio of the dose resulting in 10% surviving fraction compared to control, indicated as the reference condition.
Cell Titer-Glo survival assay
Cells were seeded to 50–70% confluence in 96-well dishes and cultured overnight prior to treatment with increasing doses of ionizing radiation. 24 hours later cells were washed once with PBS on ice then lysed directly in the well using Cell Titer-Glo reagent and incubated per manufacturer’s protocol prior to measurement of luminescence on the SpectraMax i3 plate reader. Mean relative light units (RLUs) normalized to background value of a well with no cells was plotted with standard deviation.
Time lapse confocal microscopy
Cells were seeded as described in the cell death assay except on Nunc™ Lab-Tek™ II 8-well Chambered Coverglass (155409PK, Thermo Scientific™) or 35mm cover glass culture dishes (MatTek Life Sciences, P35G-1.5-14-C), irradiated approximately 24 hours after seeding and placed on the Leica TCS SP8 X confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) beginning 24, 48, 72 or 96 hours after IR and imaged every 20–30 minutes using a 63x1.4N.A. oil objective for approximately 16 hours at a time at 37ºC in 5% CO2 using an OKO-labs cage incubator. Prior to imaging, cells were stained with LysoTracker™ Deep Red (Invitrogen™, Life Technologies Corp., Carlsbad, CA) for 15–30 minutes, washed twice with PBS and stained with propidium iodide (1:500) in FluoroBrite DMEM media (Invitrogen, Life Technologies Corp., Carlsbad, CA) with 10% FBS and 4mM L-glutamine. Images were collected using LASX software (Leica Microsystems, Buffalo Grove, IL) and visualized and analyzed using Volocity software (v6.3.5, Quorum). Image contrast and brightness were adjusted for presented images in Microsoft PowerPoint with similar adjustments for all images in the experiment (acquired with identical microscope settings). As acquired images available for review.
Transmission electron microscopy
Cells were plated in 60mm dishes to reach a confluence of ~ 70% at the time of RT, then treated 24 hours after plating with sham, 10Gy (HT3) or 30Gy (SW756) RT. Cells were harvested by trypsinization 96 hours after treatment. For ultrastructural analyses, samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 hour at room temperature. Samples were washed in sodium cacodylate buffer at room temperature and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 hour. Samples were then rinsed extensively in dH20 prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 hour. Following several rinses in dH20, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL, USA), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA).
In vivo tumor growth and radiation response
Female athymic nude mice were innoculated at 7–10 weeks of age with 0.5x106 cells suspended in a 1:1 mixture of IMDM media and Matrigel® Matrix (Corning, Glendale, AZ, USA) subcutaneously in the flank region. Tumor growth was measured by caliper twice weekly beginning at 3 days post-innoculation to determine estimated tumor volume assuming an ellipsoid sphere (V = 0.5*(L × W^2)), where V = volume, L = length of the long-axis, W = width of the short axis. Once tumors reached an appropriate size, animals were randomized to treatment with indicated doses of IR or sham IR on the SARRP as described in “Irradiation of cell lines and mice” section. Following monitored recovery from anesthesia, tumor growth was measured by caliper twice weekly until tumors reached a maximal dimension of 20mm or otherwise reached a pre-defined sacking criterion of skin ulcer.
Tumor tissue analysis by histology and immunohistochemistry
At tumor harvest, mice were sacrificed using a CO2 asphyxiation chamber and tumors immediately harvested and divided into three components for flash freezing, into 4% paraformaldehyde (PFA), and fixation buffer for TEM as described above. PFA-fixed tumor was sent and histology was performed by HistoWiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4µm. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) using standard protocols. Antigen retrieval method was heat induced epitope retrieval (HIER) at pH = 6.0 for 20 minutes (CC3) and enzyme digestion for 10 minutes (TUNEL). Antibodies used were anti-cleaved caspase-3 (Asp175) (Cell Signaling, 9661S, 1:300) and rabbit anti-rat secondary (Vector, 1:100). Bond Polymer Refine Detection Kit (Leica Biosystems, DS9800) was used according to manufacturer’s protocol. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). TUNEL staining was performed to determine TUNEL positive nuclei as a measure of fragmented nuclear DNA under standard conditions using the Promega DeadEnd Fluorometric Detection System (Promega, G3250). QuPath V0.1.2 open source software was used for digital image viewing and automated analysis using macros for nuclear staining (TUNEL) and cytoplasmic staining (CC3) to determine % cells positive. Positive control slides processed in batch under identical conditions were used to optimize macros. Full digital slide images are available at https://app.histowiz.com/shared_orders/2ccb0ce8-e4e1-41d1-8f75-a0e621ced36e/slides/.
Statistics and reproducibility
For animal experiments, based on our published and preliminary in vitro data, using a Bonferroni correction with 0.05 significance, 80% power and a two-sided t-test, we estimated needing 4 mice per group to determine an effect size of 3.5 for tumor growth kinetics. Given an anticipated tumor-take rate of 80% for HT3 and SW756 cell lines, we injected 5 mice per group for those experiments. Because all of the mice injected developed tumors, we included all in the experiment and analysis. For C33A cell line, tumor take-rate was closer to 100% so 4 mice per group were injected. Only three mice in the C33A group developed tumors. Tumor volume was estimated based on the two-dimensional caliper measurements and equation for the volume of an ellipsoid sphere: V = 0.5 * (L x W2), and means were plotted with standard error for the group. Two-way ANOVA with Bonferroni correction was performed to determine difference between mean tumor growth curves. Students’ t-test was used to compare groups in flow cytometry, cell death, TEM quantification and histology (CC3 and TUNEL) assays. Individual clonogenic survival data points were fit with the linear quadratic equation, and dose modifying factors (DMF) were calculated at 10% surviving fraction. In all cases, p-value of < 0.05 was considered significant.